Differential expression and action mechanism of lncRNA102796 in the brain of mice with chronic infection of Toxoplasma gondii

doi:10.12140/j.issn.1000-7423.2022.02.009

Abstract

Abstract:

Objective To investigate the differential expression and mechanism of long non-coding RNA102796 (lncRNA102796) in the brain of mice with chronic Toxoplasma gondii infection. Methods The model for chronic T. gondii infection was established using specific-pathogen-free female BALB/c mice. Two months after infection, the brains of infected mice (n = 10) and healthy mice (n = 10) were collected two months after infection. The expression of lncRNA102796 in mice chronically infected with T. gondii was detected by qRT-PCR, using the total RNA extracted from the brains of infected mice. The location of lncRNA102796 was determined by qRT-PCR using RNA which is extracted from the nucleus and cytoplasm of mice brains. The target gene of lncRNA102796 was predicted as opioid receptor delta 1 (oprd1) gene by bioinformatics analysis. The expression of oprd1 was further determined by qRT-PCR using the total RNA from the brains of infected mice and healthy mice. The expression of OPRD1 was further determined by Western blotting, using the brains of the infected mice. The interference and overexpression plasmids of lncRNA102796, the uninterfered lncRNA102796 plasmid pGPU6/GFP/Neo and non-overexpressed lncRNA102796 pcDNA3.1+ plasmid were used as control. The plasmids were transfected into BV-2 cells. The regulation of lncRNA102796 expression on oprd1 was investigated by qRT-PCR, using the RNA extracted from BV-2 cells. The regulation of lncRNA102796 expression on OPRD1 was investigated by western blotting, using protein extracted from BV-2 cells. The regulation of lncRNA102796 expression on cell proliferation was investigated by cell counting kit-8 assay, using BV-2 cells transfected with constructed plasmids. Constructed overexpression plasmids of anti-sense lncRNA102796 and sense lncRNA102796. The protein binding to lncRNA102796 was determined by RNA pull down and Western blotting, using the RNA and protein extracted from the overexpression BV-2 cells. Results qRT-PCR results confirmed that the expression of lncRNA102796 in the brain of mice infected chronically with T. gondii was 0.303 ± 0.054, which was down-regulated by (69.7 ± 6.7)% (t = 18.12, P < 0.05) compared to uninfected mice. The expression of lncRNA102796 was higher in the nucleus (85.04 ± 9.41)% than that in the cytoplasm (14.95 ± 9.41)% of BV-2 cells (t = 7.45, P < 0.05). The results from qRT-PCR suggested that the expression of oprd1 mRNA was 0.170 ± 0.040 in the infected mice brain, which was down-regulated (83.0 ± 5.3)% (t = 27.17, P < 0.05) compared to that of the normal mouse brain. Results from Western blotting suggested the expression of OPRD1 protein was down-regulated in the infected mice brain compared to that of normal mouse brain; Upon interfering lncRNA102796, the expression of lncRNA102796 was 0.311 ± 0.054, which was down-regulated by (68.9 ± 6.6)% compared to the control group (t = 18.00, P < 0.05); the expression of oprd1 was 0.175 ± 0.04, which was down-regulated by (82.5 ± 5.1)% compared to the control group (t = 28.08, P < 0.05); the expression of oprd1 protein also reduced. Upon overexpressing lncRNA102796, the expression of lncRNA102796 was 8.220 ± 1.192, which was up-regulated by (722.0 ± 146.0)% compared to the control group (t = 8.56, P < 0.05); the expression of oprd1 was 2.533 ± 0.365, which was up-regulated by (153.3 ± 44.7)% compared to the control group (t = 5.95, P < 0.05); the expression of oprd1 protein also increased. Results from CCK-8 assay revealed that interfered lncRNA102796 inhibited the proliferation of microglia. After 48 and 72 hours, the A₄₅₀ of BV-2 cells were 0.272 ± 0.021, 0.508 ± 0.014, respectively, which are lower than the A₄₅₀ of the control group 0.473 ± 0.024, 0.816 ± 0.014 (t = 6.35, 46.77, P < 0.05). Overexpression of lncRNA102796 can accelerate the proliferation of microglia. After 48, 72 h, the A₄₅₀ of BV-2 cells were 0.621 ± 0.038 and 1.026 ± 0.114, which were higher than the A₄₅₀ of control group 0.365 ± 0.010 and 0.530 ± 0.147 (t=10.55, P < 0.05). Results from Western blotting showed that RNA pull down confirmed that sense lncRNA102796 binds to OPRD1. Conclusion In the brain of mice chronically infected with T. gondii, lncRNA102796 suppresses microglial cell proliferation, affecting the cell cycle through binding to the target gene oprd1, thereby, causing neurological cell impairment.

Key words: Toxoplasma gondii, lncRNA102796, Neurogliocyte, Opioid receptor delta 1

CLC Number:

R531.8

WANG Zhen-xun, XIONG Si-si, SUN Xia-hui, WANG Yong-liang, PAN Ge, HE Shen-yi, CONG Hua. Differential expression and action mechanism of lncRNA102796 in the brain of mice with chronic infection of Toxoplasma gondii[J]. CHINESE JOURNAL OF PARASITOLOGY AND PARASITIC DISEASES, 2022, 40(2): 187-193.

Figures/Tables 5

References 23

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